This invention is directed to angiogenesis inhibitor compounds and methods of using these compositions to prevent and/or treat neovascularization in human patients. In particular, the compositions are useful for controlling ocular neovascularization through exogenous and endogenous therapeutic routes.
Angiogenesis is the formation of new capillary blood vessels leading to neovascularization (1). Angiogenesis is a complex process which includes a series of sequential steps including endothelial cell-mediated degradation of vascular basement membrane and interstitial matrices, migration of endothelial cells, proliferation of endothelial cells, and formation of capillary loops by endothelial cells. Though angiogenesis is a normal process for the development or maintenance of the vasculature, pathological conditions (i.e., angiogenesis dependent diseases) arise where blood vessel growth is actually harmful. Such pathologies include psoriasis, arthritis and tumor development. The progression of angiogenesis occurs in several phases which include: elaboration of the angiogenic signal; dissolution of the blood vessel basement membrane; endothelial cell proliferation; endothelial cell migration; and formation and differentiation of capillary tubules and loops. Each of these phases is a potential target for pharmacological intervention. Antiangiogenic therapy would allow modulation in such angiogenesis-associated diseases having excessive vascularization.
Angiogenesis is also associated with other important diseases of ocular tissue, including diabetic retinopathies, proliferative vitreoretinopathies and retinopathy of prematurity. Any abnormal growth of blood vessels in the eye can scatter and block the incident light prior to reaching the retina. Neovascularization can occur at almost any site in the eye and significantly alter ocular tissue function. Some of the most threatening ocular neovascular diseases are those which involve the retina. For example, many diabetic patients develop a retinopathy which is characterized by the formation of leaky, new blood vessels on the anterior surface of the retina and in the vitreous causing proliferative vitreoretinopathy. A subset of patients with age related macular degeneration develop subretinal neovascularization which leads to their eventual blindness.
The fundamental process of the formation and growth of endothelial vessels occurs during fetal development, the female endometrial cycle, wound healing, inflammation, tumor progression and tissue grafting (2, 3). In the eye, the neovascularization (de novo proliferation of endothelium and blood vessels) of ocular structures during disease or injury can disrupt ocular physiological balance and can lead to vision loss and/or blindness. Although arising from a different embryonic origin (4, 5), corneal endothelium also can undergo abnormal transdifferentiation and cause disruption of vision and blindness. Examples of visual disruption caused by ocular endothelial disfunction, proliferation and neovascularization include the retinopathies resulting as a complication from gestational prematurity, diabetes or age related macular degeneration and the iridocorneal endothelial syndromes (ICE) affecting the cornea and iris. Thus, ocular endothelia must be equipped with innate mechanisms for inhibiting excess endothelial proliferation, angiogenesis and transdifferentiation in highly specialized but relatively xe2x80x9cavascularxe2x80x9d regions such as the retina and cornea.
Several diseases involving proliferative neovascularization affect the retina and can cause visual disruption and/or blindness. The normal gradual development of a retinal blood vessel network is interrupted in retinopathy of prematurity (ROP), which results from an abnormal proliferation or neovascularization of retinal blood vessels in pregestational infants. In primary hyperplastic vitreous (PHPV), the vitreal vasculature which normally regresses late in gestation fails to regress. In ROP, retinal blood vessels which normally grow into the retinal layers in a temporally balanced manner, over-proliferate in dense patches which can lead to a range of retinal abnormalities. These defects include scarring, retinal detachment and later vision loss in those cases which do not show spontaneous regression (6-17). Abnormal vitreal vessels, which form a network between the retina and the lens, may contain subendothelial pericytes which can contract and detach the retina. To date, the only effective treatment for ROP involves ablation of the peripheral retina in an attempt to physically limit the vascular overgrowth causing the sequelae leading to later vision loss and blindness. This treatment can help to prevent blindness in ROP patients. However, retinal ablation for the ROP disease itself leaves most ROP patients with visual acuity of less than 20/40 (18).
Ocular pathology associated with diabetes mellitus ranges from retinopathy (DRO) and neovascularization of the iris to glaucoma as an end-stage complication of anterior chamber disfunction. DRO results from a twofold complication of initial retinal vascular thrombotic occlusion followed by proliferative retinal neovascularization as a result of the hypoxia caused by the vascular occlusion. The pathophysiological consequences of DRO include macular edema, ischemia and degeneration, retinal detachment, vitreous hemorrhage and optic nerve abnormalities (28). The only effective treatment for DRO is ablative therapy using lasers to photocoagulate the proliferate areas of neovascularization. However, laser therapy involves complications including retinal vein occlusion, loss of visual acuity, vitreous hemorrhage and sometimes fails altogether (20). A range of angiogenic factors and other cytokines likely contribute to neovascularization in DRO (21, 22, 23). The high serum glucose level characteristic of diabetes may itself contribute to retinal neovascularization in diabetic patients as high glucose has been shown to elevate VEGF (vascular endothelial growth factor) production from vascular smooth muscle cells (24). Both VEGF and the VEGF-R1 and VEGF-R2 are upregulated in vascular and perivascular regions of the retina in diabetic rats (25). In DRO specimens examined at stages before proliferative neovascularization peaks, VEGF is found to be expressed in retinal glial cells, retinal pigment epithelial cells and even in retinal vascular endothelial cells (26). This early production of VEGF may contribute to the later proliferative neovascularization that leads to pathological sequalae in later stages of DRO. DRO is associated with a highly abnormal local retinal microenvironment which promotes retinal neovascularization.
Macular degeneration (MDG) is the leading cause of blindness in people over age 60. The formation of a choroidal fibrovascular membrane in retinas of macular degeneration patients contributes to retinopathy and retinal detachment. Inflammatory cytokines and angiogenic growth factors including platelet derived growth factor (PDGF), acidic fibroblast growth factor (aFGF), bFGF, TGF-b1, and VEGF have been found to be present in both the retinal pigment epithelium and in the fibrovascular membranes associated with macular degeneration (26, 27). High levels of VEGF and other angiogenic cytokines are thought to lead to increased neovascularization which contributes to a positive feedback cycle of fibrovascular growth, retinal dysplasia, scarring and eventual retinal detachment.
Retinal neovascularization is often treated with multiple laser burns to the retina to remove the pathological vasculature. Patients with neovascular diseases of the anterior chamber (e.g. corneal neovascularization, iritis rubeosis) are treated with potent topical ocular glucocorticoids. These therapies are only partially effective and generally only slow neovascularization and the progress of the overall disease. In addition, they can cause severe side effects if used over a relatively long period of time.
Other attempts have been made to provide therapies for the prevention or treatment of pathological angiogenesis. For example, angiostatic steroids functioning to inhibit angiogenesis in the presence of heparin or specific heparin fragments have been described (28). Another group of angiostatic steroids useful in inhibiting angiogenesis is disclosed in commonly assigned U.S. Pat. No. 5,371,078, Clark et al., which is herein incorporated by reference.
Glucocorticoids have also been shown to inhibit angiogenesis. However, the use of glucocorticoid therapy in general is complicated by the inherent problems associated with steroid applications. Such problems include elevated intraocular pressure (29). Still other therapies have included the use of protamine (30), the use of calcitriol (31), and the use of the antibiotic, fumagillin and its analogs, disclosed in EP 354787.
Identification and characterization of new molecules regulating the formation and growth of retinal endothelium is a necessary objective for designing new therapies for controlling diseases involving retinal neovascularization. The inventors have cloned a new gene named tubedown-1 (tbdn-1), which encodes a novel protein associated with an acetyltransferase activity (32). Expression of tbdn-1 is high in developing vascular structures, including the developing vitreal vasculature, and is downregulated as tissues mature. Postnatally, tbdn-1 expression remains high in corneal, limbic, choroidal and retinal endothelia of the normal eye. Tbdn-1 is downregulated during capillary angiogenesis of IEM embryonic endothelial cells and RF/6A choroid-retina endothelial cells in vitro.
Agents which inhibit neovascularization are known by a variety of terms such as angiostatic, angiolytic, angiogenesis inhibitors or angiotropic agents.
A novel and highly conserved protein associated with an acetyltransferase activity named tubedown-1 (tbdn-1) has been isolated and characterized. Tbdn-1 regulates endothelial differentiation through protein acetylation, DNA-binding or by interacting with and/or acetylating other protein targets important for endothelial differentiation. Tbdn-1 is expressed during maturation of the developing vitreal vasculature. In normal adult eyes, tbdn-1 is expressed in the corneal endothelium proper and in the vascular endothelium of the limbus and retina. Tbdn-1 is absent or downregulated in the vascular endothelia of diseased and injured eyes, including eyes from patients with proliferative retinopathies involving neovascularization such as diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Tbdn-1 is downregulated during capillary differentiation of both IEM endothelial cells and RF/6A choroid-retina endothelial cells in vitro. Inhibition of tbdn-1 expression in IEM and RF/6A endothelial cells in vitro indicates tbdn-1 acts as an inhibitor of angiogenesis. These results taken together indicate that high levels of tbdn-1 expression present in normal ocular endothelial cells is associated with suppressing ocular neovascularization.
Accordingly, the gene tbdn-1, the cDNA of tbdn-1 (SEQ ID NO. 1), an open reading frame of tbdn-1 (such as SEQ ID NO. 6), and nucleotide sequences showing at least 70% sequence homology to SEQ ID NO. 1 or SEQ ID NO. 6, amino acid sequences translated from the cDNA of SEQ ID NO. 1, such as SEQ ID NOS. 2, 3, 4, and 5, and others amino acid sequences showing at least 85% sequence homology to SEQ ID NOS. 2, 3, 4, and 5 and which also exhibit anti-angiogenic activity may all be used as anti-angiogenic agents for treatment of ocular neovascularization. Compositions comprising a pharmaceutically effective amount of an amino acid sequence, which shows anti-angiogenic activity, that is translated from cDNA of SEQ ID NO. 1, particularly the amino acid sequences selected from the group consisting of SEQ ID NOS. 2, 3, 4 5 and a pharmaceutically acceptable carrier are also within the scope of this invention.
Methods for treating, inhibiting or delaying the onset of angiogenesis-associated diseases in mammals, wherein the angiogenesis-associated diseases are related to ocular neovascularization, are also within the scope of this invention. This method of treatment comprises treating the mammal with a pharmaceutically effective amount of an exogenously produced amino acid sequence showing anti-angiogenic activity and which is translated from the cDNA of SEQ ID NO. 1. These amino acid sequences include, but are not limited to sequences given in SEQ ID NOS. 2, 3, 4 and 5. The angiogenesis-associated diseases include, but are not limited to diabetic retinopathy, retinopathy of prematurity, primary hyperplastic vitreous, macular degeneration and any other conditions involving ocular neovascularization. The amino acid sequence may be contained in a pharmaceutically acceptable carrier and administered by intraocular injection, subretinal injection, subscleral injection, intrachoroidal injection, subconj unctival injection, topical administration or oral administration.
A gene therapy approach for treatment of mammals afflicted with an angiogeneis-associated disease, such as those related to ocular neovascularization, and in particular diabetic retinopathy and retinopathy of prematurity is also provided. For this method of treatment, an amino acid sequence, having anti-angiogenic activity, is translated from the cDNA of SEQ ID NO.1, and is provided to cells of a mammal having a deficiency in that amino acid sequence. This method further comprises administering into the cells a vector comprising and expressing a DNA sequence encoding the desired amino acid sequence, and expressing the DNA sequence in the cells to produce amino acid sequence. Cells harboring the vector secrete the amino acid sequence and this sequence is subsequently taken up by other cells deficient in the amino acid sequence. The amino acid sequences include, but are not limited to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.